Deep freezing of mouse one-cell embryos and oocytes using different cryoprotectants

THERIOGENOLOGY DEEP

FREEZING

J.J.

OF MOUSE ONE-CELL EMBRYOS AND OOCYTES DIFFERENT CRYOPROTECTANTS

Hernandez-Ledezmal'*

and R.W. Wright,

USING

Jr.3

Department of Animal Sciences Washington State University Pullman, WA 99164-6332 Received

for publication: Accepted:

hiovember 11, 1988

September 29, 1989

ABSTRACT The objective of this study was to compare iso-osmolar concentrations dimethylsulphoxide and a 1,2-propanediol, glycerol, (1.5 M) of combination of 1 M propanediol + 0.5M glycerol (PDGLY) as cryoprotectants for murine ovulated oocytes and one-cell embryos. A higher (P < DOl) percentage of one-cell embryos developed to the two-cell stage when frozen-thawed with 1,2-propanediol (83%) as compared with glycerol (43X), Data recalculated on the basis of dimethylsulfoxide (51%) or PDGLY (7%). of normal embryos thawing two-cell embryos/number one-cell after More indicated no differences among single cryoprotectant groups. in-vitrc fertilized oocytes developed to the (P < 0.01) frozen-thawed, two-cell stage when 1,2-propanedio 1 (35%) was used as cryoprotectant as Freezing-thawing resulted in a reduced compared with glycerol (15%). number of two-cell embryos after oocytes were fertilized in-vitro as compared with fresh oocytes. 1,2-propanediol was a better cryoprotectant than glycerol, dimethylsulphoxide or PDGLY for deep freezing of murine oocytes or one-cell embryos. Key words:

mouse,

embryo,

freezing,

cryoprotectants,

oocytes

INTRODUCTION Cryobiological principles used for the freezing of embryos have been The objective of these earlier studies was to applied to oocytes (1,Z). develop techniques to store mammalian embryos at low temperatures. Unfortunately, techniques for freezing oocytes have not progressed to the Difficulties in the collection, in-vitro maturation, level of embryos. in-vitro fertilization and culture of one-cell embryos, transfer to incentives have suitable recipients and perhaps a lack of economic Recent success of in-vitro limited oocyte freezing techniques. fertilization in several domestic species and humans has created new interest in the preservation of oocytes at low temperatures (3). This research was supported by Washington State University College of Agriculture and Home Economics Research Center (Project 0313). lScientific Paper No. 7985. Supported by grants from Consejo National de Ciencia y Tecnologia and Institute National de Investigaciones Forestales, Agricolas y Pecuarias 2S.A.R.H. Mexico. Centro National de Investigation Disciplinaria-Fisilogia. I.N.T.F.A.P.Postal 29-A, Dueretaro, Drd. Mexico. 3S.A.R.H. Apartado, Reprint requests.

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1989 VOL. 32 NO. 5

735

THERIOGENOLOGY

Oocytes and embryos in advanced stages of development differ both physiologically dnd morphologically (4-6). Thus freezing techniques developed for embryos may not be suitable for oocytes. However, morphologically the one-cell embryo is not too different from the recently ovulated oocyte. Therefore experience obtained from freezing one-cell embryos may be applicable to the freezing of oocytes (6). However, ultrastructural components of the oocyte may be damaged during freezing and prevent adequate development of the fertilized egg (7). It is known that spindle microtubules are disrupted if the oocytes are cooled to 0°C without the stabilizing effect of a cryoprotectant (8). Normal segregation of chromatids during completion of meiosis after sperm penetration depended on the presence of intact spindle microtubules (7). Physical studies (9) and frozen-thawed ernbryo or liposome models indicate that cryoprotectants differ in their cryoprotective ability Some cryoprotectants appear to be more toxic than others for (IO). mammalian cells (11). Oocytes have been frozen in different cryoprotecFew reports have been devoted tants (3) at different cooling rates (12). to freezing oocytes or one-cell embryos (6). Results obtained have been variable and it is not clear whether cryoprotectant differences exist for oocyte freezing. The objective of this study was to determine whether different substances provide similar cryoprotection to murine one-cell embryos or ovulated oocytes. MATERIALS Experiment

AND METHODS

1

Collection of one-cell embryos. One-cell embryos were obtained from 8 to 16-wk-old Swiss Webster females naturally bred to males of the same strain. Embryos in the cumulus cell mass were obtained from the arnpulla at noon of the day of mating. A modified Dulbecco's phosphate buffered saline solution enriched with 3 g/l bovine serum albumin (BSA) fraction V (Sigma) and 25 M of sodium lactate/l (DPBS) was used as collection medium and carrier for the cryoprotectants. Embryos were washed twice in DPBS, mechanically separated from the cumulus cell mass and pooled. One-cell embryos with morphological abnormalities or oocytes without two polar bodies were discarded. Freezing of one-cell embryos. Embryos were exposed to 1.5 M dimethylsulphoxide, glycerol, propanediol or a combination of 1.0 M propanedial + 0.5 M glycerol (PDGLY) in three steps at lo-min intervals at 23'C (12). Embryos were left in the final solution to equilibrate for 20 min before being loaded in 0.5~ml French straws. Straws were then placed in an ethanol bath freezer (FTS Systems Inc. Stone Ridge, NY) and cooled to -7'C at a rate of l"C/min. The freezing protocol has been However, instead of ending the freezing rate described previously (12). at -35"C, embryos were additionally cooled to -40°C at .l"C/min. After a 10 min equilibration at -4"C, the straws were placed in liquid nitrogen Following storage in liquia N, for 3 d, embryos were thawed in a (N ). 37'C water bath for 30 set and theLcontents were drained into sterile petri dishes. Dilution of cryoprotectants was done in six steps, decreasing the concentration of cryoprotectant by 0.25 M at 5-min intervals at 23°C.

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NOVEMBER 1969 VOL. 32 NO. 5

THERIOGENOLOGY Embryos were washed twice in DPBSA, then twice in Survival assTy. ubal Fluid medium (13) supplemented with 4 s/l BSA synthetic Human fraction V. One-cell embryos were cultured in Human Tubal Fluid droplets under paraffin oil for 24 h in a H,O saturatea 5% CO, air atmosphere at 37°C. Survival was defined as the'number of embryo& that developed to the two-cell stage 24 h after the initiation of culture. Experitnent 2 and freezing of oocytes. for Collection The same protocol addition of cryoprotectants, freezing, thawing and dilution collection, described in Experiment 1 was used in Experiment 2 with the single exception that females were not placed with males. Experiment 2a evaluated the in-vitro development to the two-cell stage of in-vitro fertilized oocytes frozen in 1.5 M of glycerol using solutions of DPBSA or Human Tubal Fluid-Hepes. Human Tubal Fluid medium (13) supplemented with 1 mg of BSA/ml was covered with paraffin oil and equilibrated at 37°C in a 5% CO., 95% air water saturated atmosphere for 24 h before addition of ooc (;I tes and sperm. Two h before oocyte thawing/sperm cells were collected by placing the vas deferens from two adult Swiss Webster males into petri dishes containing 1 ml of Human Sperm cells were forced out from Tubual Fluid covered with paraffin oil. the vas deferens by exerting pressure with two pairs of sterile watchmaker forceps. After a 30-min period, an aliquot of the suspended Concentration wa6 calculated with a semen was diluted tenfold (14). Makler counting chamber (15) and adjusted to 1 x 10 /ml (14). A 250 ~1 drop of the sperm suspension was placed in media with oocytes under paraffin oil in petri dishes. After oocytes were washed twice in Human Tubal Fluid they were added to the droplets containing the sperm suspensions and incubated for 5 to 6 h. Oocytes were then washed again in the same mediurrlwithout sperm cells and incubdted for 24 h in 50 ~1 drops of Human Tubal Fluid under paraffin oil. A control group of fresh nonfrozen oocytes was included to test the in-vitro fertilization and culture conditions. Embryos were examined for development to the two-cell stage 24 h after mixing of gametes to determine the fertilization rate. Normal two-cell embryos had a definite nucleus in each blastomere and a second polar body. Morphology of the oocytes immediately after thawing was used as a criterion for differentiating normal from degenerate oocytes. Irregular shaped oocytes with dark yellow cytoplasm were considered degenerated. Degenerated were placed in separate sperm oocytes suspension drops to determine the fertilization rate. Experiment 2b evaluated the in-vitro development to the two-cell stage of in- vitro fertilized oocytes frozen in propanediol or glycerol. Semen collection, in vitro fertilization and culture were the same as in Experiment 2a. Oocytes were frozen in glycerol or indicated propanediol in three different trials. In vitro fertilization and culture included a control group of fresh ovulated oocytes. Data from both experiments were analyzed by Chi-square.

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737

THERIOGENOLOGY Fertilized one-cell embryos (n = 30) were, as a group, each placed in 50 ~1 drops of 1.5 M DMSO, 1.5 M glycerol, 1.0 M propanediol and 0.5 glycerol and 0.5 glycerol and 1.0 M propanediol for 1 h to test for parthenogenic activation. No pathenogenic activation was observed in any of the cryoprotectant solutions tested. RESULTS Experiment 1 The effect of dimethyl sulphoxide, glycerol, propanediol and the combination propanediol-glycerol (PDGLY) on the number of one-cell embryos developing to the two-cell stage after freezing and thawing is shown in Tables 1 and 2. Frozen-thawed one-cell embryos developing to the two-cell stage as a function of the total number of embryos recovered (Table 1) showed poorer development when frozen in dimethyl sulphoxide or glycerol than in propanediol or PDGLY. Overall, propanedlol provided better (P ~0.01) protection to one-cell embryos than dimethyl sulphoxide or glycerol as evidenced by the greater number of two-cell embryos obtained after 24 h in culture. The PDGLY group had the lowest number of embryos reaching the two-cell stage (P < 0.01) of all cryoprotectant groups. To further assess the development potential of the morphological normal one-cell embryos after thawing (Hour 0) they were evaluated in relation to the number of one-cell embryos developing to the two-cell stage after 24 h in culture. A high percentage (80%) developed to the two-cell stage in all groups except for the PDGLY treatment, which was lower (P < 0.01). The number of degenerated embryos at 0 and 24 h after thawing is indicated in Table 2. As in previous tables, single cryoprotectants had a lower (P < 0.01) number of degenerated embryos at 0 h than PDGLY. No change was observed in the propanediol and PDGLY groups at 24 h. However, the number of degenerated one-cell embryos frozen in glycerol and ~~;;,"~;l sulphoxide increased significantly (P < 0.01) after 24 h in E;periment(;a; o.lo) Comparisons among frozen-thawed oocyte groups showed no dl erences However, more oocytes divided to the two-cell stage in the control group than in the frozen-thawed groups (P < 0.01). Seventy-five percent of the controls and 17% of the frozen-thawed oocytes (P < 0.01) developed to two-cell embryos within the 24 h following thawing. Experime;! 2:. Development to the two-cell stage of frozen-thawed oocytes fertl Ize in vitro after freezing in glycerol or propanediol is shown in Table 3. In general, results among trials were uniform. The total number of oocytes fertilized in vitro indicates that propanediol was a better cryoprotectant than glycerol (P < O.Ol), but freezing reduced significantly (P < 0.01) the number of two-cell embryos as compared with the unfrozen control group.

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THERIOGENOLOGY Table 1. Trial

Development in vitro of frozen-thawed one-cell embryos. Number of two-cell embryos/total recovered. d Two-cell embryoslfotfilrecovered DMSO GLY" PDC PD-GLY

1

i3/20 (65)x

2

6/16 (38)

3

12125 (48)

Total

31/61 (51)a

518 (62)% lo/26 (38)

11/13 (85)%

l/2@ ( 5)X

14/16 (87)

l/13

( 6)

18/23 (61) 23/54, (43)

43/5Zb (83)

3/45 ( 7)c

ibCDifferent superscripts per row indicate differences (PC Glycerol. "1,2-Propanedial. Table 2.

0.01).

Number of degenerated frozen-thawed one-cell embryos at 0 and 24 hours after thawing oE;,;cell embry;ih(%)

Cryoprotectant

n

Glycerol (GLY)

58

Propanediol (PD)

66

13a

(20)

17b

(25)

PD-GLY

62

33b

(52)

33a

(52)

DMSO

67

llaC (16)

gac (14)

31ad (53)

25abd(37)

Different superscripts per column (ab) or row (cd) indicate differences (PC 0.01). DISCUSSION Oocyte preservation at freezing rates lower than 0.5"C/min results in good viability and normal morphology. However, those parameters are poorly correlated with in-vitro fertilization rates in the mouse (16). The discrepancy between morphological and functional tests can be further evaluated if adequate models of similar size and shape such as one-cell embryos are subjected to the same treatments. Oocytes and one-cell embryos can be considered to be a similar physical model corresponding to a single spherical cell (6). from a morphological standpoint, results obtained from freezing one-cell embryos may be extrapolated to oocyte freezing (3). Initial studies have indicated that in-vitro matured mouse oocytes could be frozen and thawed by the use of slow freezing methods with only limited success. Perhaps the reduction observed could be due to factors not related to the cooling rate but to such factors as cryoprotectant

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THERIOGENOLOGY Table

3.

Development to oocytes fertilized

the two-cell stage of frozen-thawed mouse Comparison of cryoprotectants in-vitro. Two-cell

Trial 1

2

Controld

embryos/Oocytes Glycerol

Propanediol

18/30 (60)%

i/l1

4/17

( 91%

(24)%

i/10

5/25

13135

(20)

(37)

6141

14/36

(15)

(39)

(70) 3

inseminated

6/10 (60)

Total

12/77b (15)

ibcDifferent superscripts per row indicate In-vitro fertilized nonfrozen oocytes.

differences

31/88 (35)c (P < 0.01).

Other structural changes (18). and temperature-related toxicity researchers have suggested that cryoprotectants tnay exert toxic effects In addition, on embryos or other mammalian cell types (11, 19, 20). cryoprotectants have different cryoprotective abilities (12, 19, 20). Propanediol has been labeled as a cryoprotectant with a low tendency to form ice crystals during freezing and thawing (9) and low toxicity (11). Freezing of one-cell embryos with dimethylsulfoxide, glycerol, propanediol or PDGLY demonstrated that not all cryoprotectants are equally Propanediol was by effective when tested under similar circumstances. development to the far the best cryoprotectant (P < 0.01) for sustaining The excellent protection given two-cell stage in culture after thawing. by PDGLY to mouse blastocysts stored at low temperatures (12) could not be replicated when freezing one-cell embryos in our experiments. Results with the PDGLY group were again the lowest (P < 0.01). Data from Table 1 indicate that differences observed among cryoprotectants were due to factors inherent in the freezing process that destroyed several oocytes, and those effects could be recognized shortly after thawing in the form of degenerated embryos with disrupted cell membranes, Toxic effects of and dark yellow cytoplasm (21). irregular shape The later during the culture period. appear cryoprotectants may percentage of degenerated embryos at the initiation of culture increased significantly (P < 0.01) in glycerol and dimethylsulphoxide groups after However, no changes were observed for embryos frozen in 24 h in culture. This may indicate that glycerol and dimethylsulpropanediol or PDGLY. foxide may exert their toxic effects later in the culture period when There is sufficient embryos are not in contact with the cryoprotectant. of dimethylsulphoxide for other indicatin the toxicity evidence mammalian cells (11 4 and glycerol for embryos in more advanced stages of Prolonged exposure of mouse oocytes to development (3, 12, 19, 20, 22). dimethylsulphoxide lead to disassembling of the spindle and dispersion of the chromosomes (23).

NOVEMBER 1983 VOL.32N0.5

THERIOGENOLOGY

and DPBSA dS The Tubal Fluici-Hepes comparison of Human cryoprotectant carriers indicated that either were equally effective in sustaining development to the two-cell stage after in-vitro fertilization of frozen-thawed oocytes. fertilized frozen-thawed oocytes in-vitro The percentage of developing to two-cell embryos was higher in propanediol than in glycerol Results were similar to those obtained when (12) freezing (Table 3). Results for nonfrozen one-cell embryos (Table 1) or early blastocysts. control groups (Table 3) were always higher compared with frozen-thawed Reducea fertilization rates and an increased incidence of oocytes. chromosomdl defects in embryos derived from frozen-thawed oocytes are associated with an increase in polyploidy (3,7). However, other factors such as the surface area to volume ratio and the pertneability constant for water and temperature coefficient of that constant may hdve a role in viability (5). Osmotic studies with oocytes have reducing oocyte indicated that oocytes require longer exposure periods than embryos to and that glycerol removal needs to become fully permeated with glycerol, be reevaluated since noticeable swelling was observed in a previous study (24). Under the conditions that our experiments were carried out, propanediol was d superior cryoprotectant for one-cell embryos and oocytes stored at -196°C. The combination of 1 M propanediol + 0.5 M glycerol was not found to be a suitable cryoprotectant. REFERENCES 1.

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changes associated with e:1009-1025 (19Bl).

causes of embryo damage. bovine embryos: Vet. Learning Systems, New Jersey. (19S7). The effect the mouse

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743